Targeting of Virulence Factors and Plasmid Profiling of Klebsiella pneumoniae Causing Urinary Tract Infection in Sylhet City of Bangladesh

ABSTRACT Studies were conducted to characterize Klebsiella pneumoniae isolates from urinary tract infection (UTI) patients in Sylhet city of Bangladesh. At the same time, all isolates were screened for some common virulence genes and four significant isolates were searched for plasmid number and sizes by mini alkaline-lysis method. Among five tested isolates from female UTI patients, gyrase subunit B2 (gyrb2) amplified in all isolates, lipase and nuclease detected in three isolates and serine protease amplifies in two isolates and gave the expected band of 1130 bp, 517 bp, 1055 bp and 211 bp respectively. Two of four isolates showed 9.82 kb plasmid band on agarose gel. Isolates bearing 9.82 kb plasmid were found to be resistant to multiple commercial antibiotics. At the same time all isolates were screened for in-vitro plate assay for proteolytic, lypolytic and hemolytic activity. Isolates with positive plasmid and more than one virulent gene with gyrB2 showed positive result in in-vitro culture plate with clear zone of proteolysis, hemolysis or lipolysis. This study will be helpful for further study in finding correlation or pattern of virulence properties for K. pneumoniae associated UTI in Bangladesh.

PCR based detection of gyrB2 gene from Pseudomonas sp. affected human clinical isolates.

Detection of virulence gene is a key component in determining the pathogenicity of any isolates because these genes act multi-functionally and multi-factorially. Gyrase specific gene primer in combination of PCR technology allows precise detection of DNA gyrase subunit B2 gene (gyrB2) from different virulent microorganisms. In the present study, forward and reverse primers with a length of 20bp for both were used for detection of gyrB2 genes in clinical isolates of Pseudomonas sp. collected from patients suffering from urinary tract infection (UTI). A total of 12 isolates of Pseudomonas sp. viz., Ps1, Ps2, Ps3, Ps4, Ps5, Ps6, Ps7, Ps8, Ps9, Ps10, Ps11 and Ps12 were used in present study in which gyrB2 gene amplified in all 12 isolates and gave the expected 1130bp PCR product after visualization under gel documentation system in 1.2% agarose gel. This PCR was outstanding in the detection of gyrB2 gene in urinary tract infected patients caused by Pseudomonas sp. species.

In-vitro antibacterial activity of herbal aqueous extract against multi-drug resistant Klebsiella sp. isolated from human clinical samples.

The studies were carried out to evaluate antibacterial activity of 35 aqueous herbal extracts against a total of 20 clinical Klebshiella sp. isolates. The maximum antibacterial activity was found as 90% in crude extracts of Syzygium aromaticum (leaf) and Citrus limon L. (fruit) followed by 85% in Spondias pinnata (leaf). Sensitivity of these isolates was also evaluated for eight commercial antibiotic discs following disc diffusion assay where most of the isolates found to develop resistance against multiple commercial antibiotics. 85% of isolates exhibited resistant to chloramphenicol and erythromycin and 80% were resiatant to sulfamethoxazole and cephradine. The isolates showed their resistance between 55-60 % to the other four antibiotic discs, viz; gentamycin, streptomycin, ciprofloxacin and azithromycin. Among 35 herbal extracts tested, 19 herbal extrats were found to possess antimicrobial activity in all multi-drug resistant isolates. Therefore these herbal extracts could be used in future direction as alternative therapeutic agents for the treatment of human diseases caused by Klebsiella sp.

PCR based detection of gyrB2 gene from Pseudomonas sp. affected human clinical isolates.

Detection of virulence gene is a key component in determining the pathogenicity of any isolates because these genes act multi-functionally and multi-factorially. Gyrase specific gene primer in combination of PCR technology allows precise detection of DNA gyrase subunit B2 gene (gyrB2) from different virulent microorganisms. In the present study, forward and reverse primers with a length of 20bp for both were used for detection of gyrB2 genes in clinical isolates of Pseudomonas sp. collected from patients suffering from urinary tract infection (UTI). A total of 12 isolates of Pseudomonas sp. viz., Ps1, Ps2, Ps3, Ps4, Ps5, Ps6, Ps7, Ps8, Ps9, Ps10, Ps11 and Ps12 were used in present study in which gyrB2 gene amplified in all 12 isolates and gave the expected 1130bp PCR product after visualization under gel documentation system in 1.2% agarose gel. This PCR was outstanding in the detection of gyrB2 gene in urinary tract infected patients caused by Pseudomonas sp. species.

In-vitro antibacterial activity of herbal aqueous extract against multi-drug resistant Klebsiella sp. isolated from human clinical samples.

The studies were carried out to evaluate antibacterial activity of 35 aqueous herbal extracts against a total of 20 clinical Klebshiella sp. isolates. The maximum antibacterial activity was found as 90% in crude extracts of Syzygium aromaticum (leaf) and Citrus limon L. (fruit) followed by 85% in Spondias pinnata (leaf). Sensitivity of these isolates was also evaluated for eight commercial antibiotic discs following disc diffusion assay where most of the isolates found to develop resistance against multiple commercial antibiotics. 85% of isolates exhibited resistant to chloramphenicol and erythromycin and 80% were resiatant to sulfamethoxazole and cephradine. The isolates showed their resistance between 55-60 % to the other four antibiotic discs, viz; gentamycin, streptomycin, ciprofloxacin and azithromycin. Among 35 herbal extracts tested, 19 herbal extrats were found to possess antimicrobial activity in all multi-drug resistant isolates. Therefore these herbal extracts could be used in future direction as alternative therapeutic agents for the treatment of human diseases caused by Klebsiella sp